Synthesis, Characterization, and Antiproliferative Properties of New Bio-Inspired Xanthylium Derivatives

Xanthylium derivatives are curcumin analogs showing photochromic properties. Similarly, to anthocyanins, they follow the same multistate network of chemical species that are reversibly interconverted by external stimuli. In the present work, two new asymmetric monocarbonyl analogues of curcumin, 4-(4-hydroxy-3-metoxybenzylidene)-1,2,3,4-tetrahydroxanthylium chloride (compound 3) and 4-(4-hydroxybenzylidene)-6-methoxy-1,2,3,4-tetrahydroxanthylium chloride (compound 4) were synthesized, and their photochromic and biological properties were investigated. The UV-Vis spectroscopy and the direct and reverse pH-jumps studies confirmed the halochromic properties and the existence of different molecular species. A network of chemical reactions of these species was proposed. Furthermore, the antiproliferative properties of both compounds were evaluated using P19 murine embryocarcinoma cells and compared with each other. The results demonstrate that both new xanthylium derivatives modify the progression through the cell cycle of P19 cells, which translates into a significant antiproliferative effect. The effect of the methoxy group position is discussed and several checkpoint proteins are advanced as putative targets.


Introduction
Plants and plant extracts were from ancient times essential sources of bioactive compounds, particularly drugs, also becoming an important part of the medicine of our days [1,2]. The discovery of such bioactive molecules by screening and isolation from natural sources was accompanied by synthesis of derivatives or structural analogues, which also emerged as drug candidates, sometimes with even enhanced pharmacological effect especially for cancer and infectious diseases. These developments revitalized the interest for natural products as drug leads [3].
Among the bioactive components of plants, polyphenols are a diverse and multifunctional group with substantial health potential, including cancer prevention and treatment [4]. These health-promoting effects are linked to their antioxidant activity, leading to important antibiotic, anticancer, and anti-inflammatory properties. Polyphenols and their synthetic analogs interfere in carcinogenesis by modulating and regulating multiple signaling pathways and transcription factors, membrane-associated receptor tyrosine kinases, fatty acid metabolism and lipid rafts or methylation, together with other emerging targets [5]. Various synthetic analogues of natural polyphenols demonstrated strong anticancer activity, including (-)-epigallocatechin-3-gallate analogues [6], synthetic anthocyanidins [7], or cinnamic acid derivatives [8]. Some analogues with known anticarcinogenic effect

UV-Vis and FT-IR Spectroscopy
UV-Vis absorption spectra were registered at 20 • C using an Agilent Cary 60 spectrophotometer (Agilent Technologies, Waldbronn, Germany).
FT-IR spectra were collected on a Bruker Vertex 70 (Bruker Daltonik GmbH, Bremen, Germany) spectrometer connected with a Platinium ATR, Bruker Diamond Type A225/Q. The samples spectra were collected after 64 co-added scans, on a spectral domain of 4000-400 cm −1 , with a resolution of 4 cm −1 .

NMR Analysis
NMR spectra were collected on a Bruker AVANCE III spectrometer (Bruker Daltonik GmbH, Bremen, Germany) working at 500.0 MHz ( 1 H) and 125.0 MHz ( 13 C) at 298 K. Chemical shifts δ are reported in ppm versus tetramethylsilane, TMS, coupling constants are reported in Hz and for splitting pattern the following abbreviations are used: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets) and m (multiplet). The samples were dissolved in DMSO-d 6 or CD 3 OD. NMR assignments have been performed based on 1D NMR spectra ( 1 H, 13 C, DEPT 135) and 2D NMR spectra (COSY, HQSC, HMBC) analysis.

Study of pH Dependent Photochromic Properties
The halochromic properties study of the synthesized xanthylium dyes involved spectrophotometric monitoring of color variation of dye solutions at different pH values over time. The buffer solutions in the pH range from 2 to 12 were prepared based on boric acid, citric acid and trisodium phosphate solutions following a previously described procedure [27].

Synthesis and Characterization of the Xanthylium Dyes
Two new synthetic bio-inspired xanthylium salts, obtained in a closed form, were synthesized, and characterized. Figure 1 depicts the reaction scheme for the synthesis of compounds 3 and 4.

Study of pH Dependent Photochromic Properties
The halochromic properties study of the synthesized xanthylium dyes involved spectrophotometric monitoring of color variation of dye solutions at different pH values over time. The buffer solutions in the pH range from 2 to 12 were prepared based on boric acid, citric acid and trisodium phosphate solutions following a previously described procedure [27].

Biological Activity Study
The P19 cells were cultured at 37 °C and 5% CO2 atmosphere in a SANYO MCO-5AC incubator. Cell quantification and cell cycle was studied with the Muse Cell Analyzer microflow-cytometer using dedicated reagent kits: Muse System Check Kit (MCH100101) for device calibration, Muse Count and Viability Kit (MCH100102) for live cell counting and Muse Cell Cycle Kit (MCH100106) for aggregate cell cycle analysis.

Synthesis and Characterization of the Xanthylium Dyes
Two new synthetic bio-inspired xanthylium salts, obtained in a closed form, were synthesized, and characterized. Figure 1 depicts the reaction scheme for the synthesis of compounds 3 and 4. The structure and the purity of the synthesized salts were assessed and demonstrated based on FT-IR, 1D and 2D NMR spectra.
The FT-IR analysis confirmed the presence of the main functional and structural groups in all the synthesized compounds as presented inSection 2.2.1.
The exact structures of the synthesized dyes were demonstrated by NMR analysis. In the 1 H-NMR spectrum of compound 3, the multiplets at 2.07-2.02 ppm, 3.12-3.09 ppm and the signals at 3.14 and 3.92 ppm, which correspond to aliphatic protons were attributed to the methylene protons from the cyclohexanone moiety (-CH2) and to the methoxy group (-OCH3) from vanillin. The aromatic protons showed signals between 6.95 and 8.19 ppm. The most deshielded signals were found for protons in CH groups due to the extended π conjugation of the molecule (H7 and H14, Supplementary Materials, Figure S5). The signals at 22.0, 28.8, and 29.4 ppm in the 13 C-NMR spectrum of compound 3 correspond to the carbon atoms of the three methylene groups (-CH2) present in the structure. Another aliphatic signal was identified at 56.8 ppm and attributed to the carbon atom from the methoxy group (-OCH3). The aromatic carbon atoms were assigned to The structure and the purity of the synthesized salts were assessed and demonstrated based on FT-IR, 1D and 2D NMR spectra.
The FT-IR analysis confirmed the presence of the main functional and structural groups in all the synthesized compounds as presented in Section 2.2.1.
The exact structures of the synthesized dyes were demonstrated by NMR analysis. In the 1 H-NMR spectrum of compound 3, the multiplets at 2.07-2.02 ppm, 3.12-3.09 ppm and the signals at 3.14 and 3.92 ppm, which correspond to aliphatic protons were attributed to the methylene protons from the cyclohexanone moiety (-CH 2 ) and to the methoxy group (-OCH 3 ) from vanillin. The aromatic protons showed signals between 6.95 and 8.19 ppm. The most deshielded signals were found for protons in CH groups due to the extended π conjugation of the molecule (H7 and H14, Supplementary Materials, Figure S5). The signals at 22.0, 28.8, and 29.4 ppm in the 13 C-NMR spectrum of compound 3 correspond to the carbon atoms of the three methylene groups (-CH 2 ) present in the structure. Another aliphatic signal was identified at 56.8 ppm and attributed to the carbon atom from the methoxy group (-OCH 3 ). The aromatic carbon atoms were assigned to signals between 117.6 and 156.6 ppm. The carbon atoms linked to oxygen atoms were found to be the most deshielded ones with signals at 174.1 ppm (C=O + ), 156.6 ppm (C-O + ), 154.8 (C-OH), 149.7 ppm (C-OCH 3 ). Additional proof of the formation of compound 3 was given by the remote couplings between carbon atoms and protons, shown in the two-dimensional HMBC spectrum ( Figure 2). The signal corresponding to C1 carbon atom (174.5 ppm) coupled over two bonds with protons H3 (3.14 ppm), H5 (3.12-3.09 ppm), H7 (8.63 ppm) and H14 (8.78 ppm). It can also be observed the remote coupling of C10 (149.7 ppm) with three protons: H23 (3.92 ppm), H9 (7.44 ppm) and H12 (6.95 ppm). The remote coupling over two bonds of C14 carbon atom (151.4 ppm) with protons H5 (3.12-3.09 ppm) and H16 (8.09 ppm) was a further confirmation of the formation of compound 3.
Since the only structural difference between compounds 3 and 4 is the position of the methoxy group for compound 4 the characterization details are presented in the Supplementary Materials.

pH-Dependent Photochromic Properties
The halochromic properties of the synthesized salts were investigated by UV-Vis spectroscopy.
Similar to the anthocyanins, these xanthylium derivatives show photochromic properties and follow the same multistate network of chemical species, which can reversibly pass from one species to another when are subjected to the action of different stimuli like pH changes, temperature, or light [20].  Since the only structural difference between compounds 3 and 4 is the position of the methoxy group for compound 4 the characterization details are presented in the Supplementary Materials.

pH-Dependent Photochromic Properties
The halochromic properties of the synthesized salts were investigated by UV-Vis spectroscopy.
Similar to the anthocyanins, these xanthylium derivatives show photochromic properties and follow the same multistate network of chemical species, which can reversibly pass from one species to another when are subjected to the action of different stimuli like pH changes, temperature, or light [20]. Figure 3 depicts a potential network of chemical reactions involved in the interconversion of the species under acidic and basic conditions. The existence of these species was confirmed by the UV-Vis spectra (Figures 4 and 5). It should be noted that the structural differences between compounds 3 and 4, namely the different positioning of the methoxy group on the aromatic ring, did not significantly influence the halochromic properties and the corresponding species exhibit almost similar absorption bands. Molecules 2023, 28, x FOR PEER REVIEW 7 of 16 The existence of these species was confirmed by the UV-Vis spectra (Figures 4 and 5). It should be noted that the structural differences between compounds 3 and 4, namely the different positioning of the methoxy group on the aromatic ring, did not significantly influence the halochromic properties and the corresponding species exhibit almost similar absorption bands.
Since the synthesis of compounds 3 and 4 was accomplished in acidic medium, the purple-colored xanthylium cations AH + (compound 3: λ = 539 nm; compound 4: λ = 521 nm) were obtained. The AH + species were transformed into blue-colored quinoidal bases (A) (compound 3: λ = 624 nm with two shoulders around 586 nm and 670 nm; compound 4: λ = 625 nm), by a proton transfer process. When hydrating the AH + cations, colorless hemiketals (B) are obtained, which upon increasing the pH, by opening the cycle, convert to the cis and trans chalcones (Cct, Ctt), yellow-colored neutral species (compound 3: λ = 345 nm; compound 4: λ = 363 nm). The orange-colored ionized species (Ctt 2− ) (compound 3: λ = 459 nm; compound 4: λ = 464 nm) are obtained by deprotonation of the hydroxyl groups in basic medium. Figures 4 and 5 show the absorption spectra of compounds 3 and 4, collected after 40 min and 72 h at different pH values. It must be specified that the AH + species corresponding to the strong acid medium (pH = 2) and those corresponding to strong basic medium Ctt 2− (pH = 12) are stable in time at room temperature, so that they can be stored for long periods of time.   The existence of these species was confirmed by the UV-Vis spectra (Figures 4 and  5). It should be noted that the structural differences between compounds 3 and 4, namely the different positioning of the methoxy group on the aromatic ring, did not significantly influence the halochromic properties and the corresponding species exhibit almost similar absorption bands.
Since the synthesis of compounds 3 and 4 was accomplished in acidic medium, the purple-colored xanthylium cations AH + (compound 3: λ = 539 nm; compound 4: λ = 521 nm) were obtained. The AH + species were transformed into blue-colored quinoidal bases (A) (compound 3: λ = 624 nm with two shoulders around 586 nm and 670 nm; compound 4: λ = 625 nm), by a proton transfer process. When hydrating the AH + cations, colorless hemiketals (B) are obtained, which upon increasing the pH, by opening the cycle, convert to the cis and trans chalcones (Cct, Ctt), yellow-colored neutral species (compound 3: λ = 345 nm; compound 4: λ = 363 nm). The orange-colored ionized species (Ctt 2− ) (compound 3: λ = 459 nm; compound 4: λ = 464 nm) are obtained by deprotonation of the hydroxyl groups in basic medium. Figures 4 and 5 show the absorption spectra of compounds 3 and 4, collected after 40 min and 72 h at different pH values. It must be specified that the AH + species corresponding to the strong acid medium (pH = 2) and those corresponding to strong basic medium Ctt 2− (pH = 12) are stable in time at room temperature, so that they can be stored for long periods of time.  In the case of compound 3, in the acidic pH domain (2)(3)(4) increasing the pH leads to the bleaching of the solutions in time, corresponding to the formation of a hemiketal (B). In the pH range 5 to 6, the predominant species is the quinoidal base (A). At pH values 7 and 8, the formation of the neutral species Cct, Ctt can be observed ( Figure 6). The deprotonated forms of the neutral species appear in the pH range 9 to 12. The quinoidal base (A) was observed only until the pH reached the value 10, above which the solutions become orange and exhibit two absorption bands at 345 nm and 459 nm, corresponding to the neutral and deprotonated species. hemiketals (B) are obtained, which upon increasing the pH, by opening the cycle, convert to the cis and trans chalcones (Cct, Ctt), yellow-colored neutral species (compound 3: λ = 345 nm; compound 4: λ = 363 nm). The orange-colored ionized species (Ctt 2− ) (compound 3: λ = 459 nm; compound 4: λ = 464 nm) are obtained by deprotonation of the hydroxyl groups in basic medium. Figures 4 and 5 show the absorption spectra of compounds 3 and 4, collected after 40 min and 72 h at different pH values. It must be specified that the AH + species corresponding to the strong acid medium (pH = 2) and those corresponding to strong basic medium Ctt 2− (pH = 12) are stable in time at room temperature, so that they can be stored for long periods of time.
In the case of compound 3, in the acidic pH domain (2)(3)(4) increasing the pH leads to the bleaching of the solutions in time, corresponding to the formation of a hemiketal (B). In the pH range 5 to 6, the predominant species is the quinoidal base (A). At pH values 7 and 8, the formation of the neutral species Cct, Ctt can be observed ( Figure 6). The deprotonated forms of the neutral species appear in the pH range 9 to 12. The quinoidal base (A) was observed only until the pH reached the value 10, above which the solutions become orange and exhibit two absorption bands at 345 nm and 459 nm, corresponding to the neutral and deprotonated species.  In the pH range 3 to 6, compound 4 is not stable in time and bleaching of the solutions was observed, corresponding to the formation of the hemiketal (B). In the pH range 7 to 9 the predominant species are the quinoidal base (A) and cis and trans chalcones (Cct, Ctt), which are not stable over time.
The transformations of species for compound 4 over time are shown in Figure 7. At pH 8, the formation of neutral species Cct, Ctt from the quinoid base (A) was noticed, while at pH 9 the formation of the species (Ctt 2− ) was observed. In the pH range 3 to 6, compound 4 is not stable in time and bleaching of the solutions was observed, corresponding to the formation of the hemiketal (B). In the pH range 7 to 9 the predominant species are the quinoidal base (A) and cis and trans chalcones (Cct, Ctt), which are not stable over time.
The transformations of species for compound 4 over time are shown in Figure 7. At pH 8, the formation of neutral species Cct, Ctt from the quinoid base (A) was noticed, while at pH 9 the formation of the species (Ctt 2− ) was observed.

pH-Jumps Study
To investigate the reversibility of the multistate network of species, direct and reverse pH-jumps were performed. A direct pH-jump involves the addition of base to solutions equilibrated in a strongly acidic medium, while a reverse pH-jump corresponds to the addition of acid to solutions stable at higher pH values.
Upon a direct pH-jump from an equilibrated solution of compound 3, at pH = 2.07 to pH = 12.13, only the formation of the deprotonated species was observed (Figure 8a). The transition from the AH + species to the quinoidal base (A) is achieved through a proton transfer, which is the fastest kinetic process that occurs and can be captured spectrophotometrically only with a stopped-flow system. The spectral variations accompanying a reverse pH-jump of an equilibrated solution of compound 3 from pH = 12.06 to pH = 7.23 leading to the formation of the neutral species cis and trans chalcones (Cct, Ctt) and the quinoidal base (A) are shown in Figure 8b).
The formation of the deprotonated species (Ctt 2− , λ = 459 nm) from the quinoidal base (A, λ = 624 nm with two shoulders around 586 nm and 670 nm) was evidenced also by the direct pH-jump of a solution of compound 3 equilibrated from pH 6.09 to 12.16 ( Figure 9).

pH-Jumps Study
To investigate the reversibility of the multistate network of species, direct and reverse pH-jumps were performed. A direct pH-jump involves the addition of base to solutions equilibrated in a strongly acidic medium, while a reverse pH-jump corresponds to the addition of acid to solutions stable at higher pH values.
Upon a direct pH-jump from an equilibrated solution of compound 3, at pH = 2.07 to pH = 12.13, only the formation of the deprotonated species was observed (Figure 8a). The transition from the AH + species to the quinoidal base (A) is achieved through a proton transfer, which is the fastest kinetic process that occurs and can be captured spectrophotometrically only with a stopped-flow system.

pH-Jumps Study
To investigate the reversibility of the multistate network of species, direct and reverse pH-jumps were performed. A direct pH-jump involves the addition of base to solutions equilibrated in a strongly acidic medium, while a reverse pH-jump corresponds to the addition of acid to solutions stable at higher pH values.
Upon a direct pH-jump from an equilibrated solution of compound 3, at pH = 2.07 to pH = 12.13, only the formation of the deprotonated species was observed (Figure 8a). The transition from the AH + species to the quinoidal base (A) is achieved through a proton transfer, which is the fastest kinetic process that occurs and can be captured spectrophotometrically only with a stopped-flow system. The spectral variations accompanying a reverse pH-jump of an equilibrated solution of compound 3 from pH = 12.06 to pH = 7.23 leading to the formation of the neutral species cis and trans chalcones (Cct, Ctt) and the quinoidal base (A) are shown in Figure 8b).
The formation of the deprotonated species (Ctt 2− , λ = 459 nm) from the quinoidal base (A, λ = 624 nm with two shoulders around 586 nm and 670 nm) was evidenced also by the direct pH-jump of a solution of compound 3 equilibrated from pH 6.09 to 12.16 (Figure 9). The spectral variations accompanying a reverse pH-jump of an equilibrated solution of compound 3 from pH = 12.06 to pH = 7.23 leading to the formation of the neutral species cis and trans chalcones (Cct, Ctt) and the quinoidal base (A) are shown in Figure 8b).
The formation of the deprotonated species (Ctt 2− , λ = 459 nm) from the quinoidal base (A, λ = 624 nm with two shoulders around 586 nm and 670 nm) was evidenced also by the direct pH-jump of a solution of compound 3 equilibrated from pH 6.09 to 12.16 (Figure 9). Molecules 2023, 28, x FOR PEER REVIEW 10 of 16

Inhibition of Cell Proliferation
The characterization of the biological activity of the compounds 3 and 4 was performed on P19 embryonic teratocarcinoma cells and involved the determination of the antiproliferative concentrations and the effect on cell cycle progression. P19 cells are frequently used for the evaluation of cytotoxicity and the impact on the cell cycle of synthetic or natural compounds [28,29], and were adopted it in our study as an ex vivo model for screening for antiproliferative effects, due to their ease of maintenance in culture and high proliferation rate.

Inhibition of Cell Proliferation
The characterization of the biological activity of the compounds 3 and 4 was performed on P19 embryonic teratocarcinoma cells and involved the determination of the antiproliferative concentrations and the effect on cell cycle progression. P19 cells are frequently used for the evaluation of cytotoxicity and the impact on the cell cycle of synthetic or natural compounds [28,29], and were adopted it in our study as an ex vivo model for screening for antiproliferative effects, due to their ease of maintenance in culture and high proliferation rate.

Inhibition of Cell Proliferation
The characterization of the biological activity of the compounds 3 and 4 was performed on P19 embryonic teratocarcinoma cells and involved the determination of the antiproliferative concentrations and the effect on cell cycle progression. P19 cells are frequently used for the evaluation of cytotoxicity and the impact on the cell cycle of synthetic or natural compounds [28,29], and were adopted it in our study as an ex vivo model for screening for antiproliferative effects, due to their ease of maintenance in culture and high proliferation rate. Table 1 shows the arithmetic mean, the final number of cells, the statistical significance of the obtained results, and the percentage of proliferation inhibition for the two compounds. Table 1. Dose-dependent inhibition of P19 cells exposed to compounds 3 and 4 (Student t-test, 2 tails, heteroscedastic). Compound 3 strongly inhibits P19 cells proliferation starting with concentrations above 100 µM (statistical significance threshold p = 0.05). Similarly, compound 4 also inhibits P19 cells proliferation starting with concentrations above 50 µM (statistical significance threshold p = 0.05). The ostensibly more pronounced antiproliferative effect of compound 4 compared to 3 might be due to the different positioning of the methoxy group ( Figure 13).

The Effect on Cell Cycle Progression
To further dissect the biological activity of the new xanthylium derivatives, their influence on cell cycle progression were analyzed, selecting two concentrations close to IC 50 values and at which the proliferation inhibition effect is comparable (around 50%): 200 µM for compound 3, and 100 µM for compound 4. Table 2 shows the percentage of P19 cells in each phase of the cell cycle and the analysis of statistical significance. Compound 3 produces a significant accumulation of the cell population in the G2/M phase (with a corresponding decrease of distribution in G0/G1 and S phases), indicating a possible blockage of progression to or through mitosis by action at the level of G2 and/or M checkpoints.
Compound 3 strongly inhibits P19 cells proliferation starting with concentrations above 100 µM (statistical significance threshold p = 0.05). Similarly, compound 4 also inhibits P19 cells proliferation starting with concentrations above 50 µM (statistical significance threshold p = 0.05). The ostensibly more pronounced antiproliferative effect of compound 4 compared to 3 might be due to the different positioning of the methoxy group ( Figure 13). Figure 13. Dose-dependent inhibition of P19 cells exposed to compounds 3 (blue) and 4 (red).
Poly (3): y = −9 × 10 −11 x 4 + 3 × 10 −7 x 3 -0.0004x 2 + 0.3155x + 0.5655; R² = 0.9955 Poly (4): y = −5 × 10 −10 x 4 + 1 × 10 −6 x 3 -0.0014x 2 + 0.6012x + 8.4036; R² = 0.9826 Figure 13. Dose-dependent inhibition of P19 cells exposed to compounds 3 (blue) and 4 (red). As expected, the P19 cells exposed to compound 4 show a similar cell cycle distribution profile with a significant accumulation in G2/M phase and a decrease of the number cells in G0/G1 and S phases. This suggests that compound 4 also impacts the activity of the proteins involved in G2 and M checkpoints (Cyclin A, Cyclin B, CDK1, etc.). Although the cell cycle redistribution in G2/M phase is similar for the two compounds, there are significant changes in G0/G1 and S phase cell numbers. Nevertheless, the overall distribution is similar, with preponderance of cells in the G2/M phase, indicating similar mechanisms of action.
The mechanisms underlying these effects involve most probably impact on the control of the G2/M checkpoint and on the pro-apoptotic mechanisms. From this perspective, these results are well in line with published data exploring antiproliferative and pro-apoptotic effects of curcumin derivatives on cancer cells [14]. Vero et al. [31] described a strong cytotoxic effect of similar asymmetrical mono-carbonyl curcumin analogues on Vero, HeLa, and MCF7 cells, with LC 50 and IC 50 values in the micromolar range similar to the ones determined in our experiments. Qiu et al. [12] reported a significant, dose-dependent G2/M arrest and apoptosis of cholangiocarcinoma cell lines exposed to allylated monocarbonyl curcumin analogues presumably through p53/Cdc2 and Bax/Bcl-2 signaling, respectively. Similar effects were described by Xia et al. [32] on human gastric cancer cell lines exposed to asymmetric mono-carbonyl analog of curcumin. Overall, it appears that the G2/M blockage and induction of apoptosis are general features of curcumin derivatives, the variation in biological activities being given by the nature and position of the different side groups.

Conclusions
Two new synthetic bio-inspired xanthylium salts were synthesized and fully characterized. By performing a UV-Vis spectroscopy study, and direct and reverse pH-jumps, the halochromic properties and the reversibility of the multistate network of species were explored. A network of chemical reactions of the species was also proposed.
Both compounds 3 and 4 have a significant biological antiproliferative activity on P19 embryocarcinoma cells, with IC 50 values around 100 µM for compound 4 and 200 µM for compound 3. For the time being, we do not know whether this effect is due only to the inhibition of cell cycle progression or it also associates a pro-apoptotic effect.
The position of the methoxy group does not seem to alter the overall biological effect of the two xanthylium derivatives: the inhibition of cell proliferation through blockage of G2/M progression. Nevertheless, the different positions of the methoxy group might be responsible for the changes observed in G0/G1 and S phase distribution. Furthermore, the position of the methoxy group might also significantly influence the compound concentration at which the inhibitory effect becomes significant: 50 µM for compound 4 and 100 µM for compound 3.
Further experiments will be performed to elucidate the interactions of these molecules, the role of positioning the methoxy group in benzene cycles in modulating these interactions and the spectrum of intracellular signaling changes caused by exposure to these molecules.